This invention relates to the transfer of liquids from one vessel to another. In particular, the invention relates to an improved method of transferring small quantities of liquid from a plurality of wells to a plurality of receptacles.
Continuing rapid advances in chemistry, particularly in biochemistry and molecular biology, demand improved capabilities for carrying out large numbers of reactions using small quantities of materials.
In screening patients for genetic disease and susceptibility, for example, the number of conditions for which associated mutations are known is growing, and the numbers of mutant alleles known to be associated with these conditions is increasing. An adequate genetic screen for one or even a few of these conditions can require testing a sample from the patient against a very large number of genetic probes.
Enormous and rapidly increasing numbers of critical biomolecules have been identified and characterized, and an understanding of their various roles in cellular processes is vastly improving. Consequently, for example, the number of potential targets for pharmacologic intervention is very large. Techniques for parallel chemical synthesis, such as combinatorial chemistries, can efficiently produce libraries of large numbers of synthetic compounds that may be screened against selected targets in a rational drug design approach.
Considerable effort has been directed to developing better approaches to handling large numbers of samples, reagents, and analytes. Automated laboratory workstations and robotics-based systems have been brought to routine use for some chemical manipulations in screening and synthesis, and dedicated computer applications have been developed both for controlling processes and for manipulating data. And a number of approaches have been proposed for miniaturizing systems for carrying out chemical processes, to reduce the quantities of the various components. Some of these approaches have found use. Particularly, for example, array technologies for binding pair assays use components immobilized in arrays of features on a surface, and microfluidics technologies employ networks of interconnected capillaries to move and combine components on a very small scale.
There is significant and growing interest in employing array technologies for conducting biomolecular manipulations. In array techniques certain of the components are immobilized in a pattern of array features on a surface of a solid support, and permitted to interact with other components. Arrays of binding agents, in which such binding agents as oligonucleotides or peptides are deposited onto a support surface in the form of an array or pattern, can be useful in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. For example, information about the nucleotide sequence of a target nucleic acid may be obtained by contacting the target with an array of different surface-bound DNA probes under conditions that favor hybridization of nucleic acids having complementary sequences, and determining at what sites on the array duplexes are formed. Hybridization to surface-bound DNA probe arrays can provide a relatively large amount of information in a single experiment. And, for example, array technology can be useful in differential gene expression analysis.
Such arrays may be prepared in any of a variety of different ways. For example, DNA arrays may be prepared manually by spotting DNA onto the surface of a substrate with a micropipette. See, Khrapko et al., DNA Sequence (1991), 1:375-388. Or, a dot-blot approach or a slot-blot approach may be employed in which a vacuum manifold transfers aqueous DNA samples from a plurality of wells to a substrate surface. Or, an array of pins can be dipped into an array of fluid samples and then contacted with the substrate to produce the array of sample materials. Or, an array of capillaries can be used to produce biopolymeric arrays, as described for example in International Patent Publication WO 95/35505.
U.S. patent application Ser. Nos. 09/150,504 and 09/150,507 describe forming biomolecular arrays by adaptations of devices employed in the printing industry and, particularly, of inkjet print heads and of automated devices for moving a print head over a print surface and for depositing the inks at desired locations on the surface. These references and others cited herein, above and below, are incorporated herein in their entirety by reference. Other uses of inkjet printing devices to dispense biochemical agents, such as proteins and nucleic acids, are suggested or disclosed in, for example, U.S. Pat. Nos. 5,658,802; 5,338,688; 5,700,637; 5,474,796; 4,877,745; and 5,499,754.
Whether the miniaturized system is a microfluidic device or an array, or is of some other design, at least some of the various biomolecules to be introduced to the system are typically prepared in depots remote from the receptacles by which they are introduced to the system. These depots may take the form of a multiwell plate (conventionally providing 96 wells in a 12xc3x978 format), for example, or a microtiter plate (conventionally providing 384 wells in a 16xc3x9724 format, or 1536 wells in a 32xc3x9748 format). A technical challenge is presented by the step of transferring the liquids containing the various biomolecules from the depots to the specific receptacles. In an array system constructed using an inkjet printing technique, for example, a technical challenge is presented by the need to transfer the liquids from the depots to the specific reservoirs in the print head.
Conventionally a pipette may be employed to transfer a liquid dropwise from a depot to a receptacle (such as a reservoir in a microfluidics device or a reservoir in a print head). The tip of the pipette is first dipped into the liquid in the depot and some of the liquid is drawn into the pipette; then the pipette is moved to the receptacle and a quantity of the liquid is expelled into the receptacle. Several pipettes may be ganged and used to transfer several different liquids at once, to reduce the number of repetitions, but problems of small dimension may make such an approach impractical. In any event the transfer step results in contamination of pipettes, which accordingly must be either discarded and replaced or decontaminated (for example by rinsing) before they are used to transfer different liquids. Where a large number of different liquids are to be moved, the: transfer apparatus become mechanically unwieldy, and the cost of minimizing the risk of contamination is increased.
Co-pending patent application Ser. No. 09/183,604 provides a method and apparatus for liquid transfer that overcomes the problems in the art with liquid transfer of a small quantity of a sample. The method and apparatus can be applied to inkjet print head technology. Different liquid samples are stored in depots or wells in a plate or block, such as for example, in a standard microtiter plate. The samples may be biological materials that are used in analytical assays, such as on array assays, for example. The samples are transferred (loaded) into corresponding receptacles of a receiving system. The samples are held in the receptacles until dispensed for an assay. According to co-pending application Ser. No. 09/183,604, the liquid sample is caused to move out of the depots on the microtiter plate and form a droplet with a convex meniscus at the surface of each depot. The receiving system extracts or loads the droplet into its receptacles by contacting the openings in the receptacles with the menisci of the liquid samples. The flow of the liquid sample into the receptacle (loading) relies at least in part on capillary action.
The loading efficiency for the method and apparatus of the co-pending application is difficult to determine until the sample is then dispensed or fired on the test specimen by the receiving system. If a sample did not load properly or completely into the receptacle, then the receptacle will not fire properly. For example, either no sample will be dispensed or an insufficient amount will be dispensed onto the array substrate. The firing reliability of a single receptacle of the co-pending apparatus and method is estimated to be approximately 75%. The efficiency and reliability of the receiving system directly impact the reliability of the analytical results of biomolecular assays.
Thus, it would be advantageous to improve the loading efficiency and therefore, the firing reliability of the above liquid transfer apparatus and method. Such an improvement would increase the reliability of biological assays.
The present invention provides a method and apparatus of liquid transfer with improved loading efficiency and therefore, improved firing reliability relative to the co-pending apparatus and method.
In one aspect of the invention, an apparatus for transferring one or more liquid samples is provided. The apparatus comprises a depot member and a receiving member. The depot member has a plurality of wells and the receiving member has at least one receptacle and a plurality of orifices. The plurality of orifices is in fluid communication with the receptacle. Each well in the depot member has an opening at one surface of the depot member. The wells support the one or more liquid samples. The liquid sample is displaced from the well through the opening as a spherical droplet at the surface of the depot member for transfer. The apparatus further comprises a controller for aligning the plurality of orifices of the receiving member over the displaced droplet on the surface of the depot member. The controller further lowers the receiving member to a height above the surface of the depot member to contact and to compress the droplet. Preferably, the droplet is compressed until the droplet contacts all of the orifices in the receiving member. Thereafter, the controller preferably activates back pressure to load the compressed droplet through essentially all of the contacted orifices. Advantageously, the apparatus has reduced sensitivity to positioning errors since the compressed droplet contacts all of the orifices of the receptacle prior to loading into the receiving member.
In another aspect of the invention, a method of transferring liquid samples is provided. The method transfers the liquid samples from a plurality of wells into at least one receptacle in the receiving member through orifices in fluid communication with the receptacle. Each liquid sample has a droplet shape when displaced from the plurality of wells. The method comprises the steps of compressing the displaced droplet with the receiving member. Preferably, the droplet is compressed until it contacts all of the orifices. The method further comprises the step of activating back pressure to load the compressed droplet into the receptacle through essentially all of the contacted orifices.
The receiving member advantageously may have a subsequent firing reliability of at least approximately 95% using the method of the invention, because the liquid sample is spread to contact all of the orifices of the receiving member during the compression step.
In a preferred embodiment, the receiving member is an inkjet print head adapted for transfer of biological samples in analytical assays. The print head has at least one reservoir in fluid communication with a plurality of nozzles. The print head is lowered over the spherical liquid droplet to compress the droplet until the droplet contacts all of the nozzles of the plurality of nozzles. When the liquid samples are the same in each well of the depot member, the print head efficiently transfers the same liquid samples from the plurality of wells of the depot member to different locations on an array substrate, for example. When the liquid samples are different in each well of the depot member, the print head has a plurality of separate reservoirs, each with its own plurality of nozzles, to efficiently transfer the different liquid samples from the plurality of wells to different or the same locations on the array substrate.
The efficiency of the apparatus and method provides for more reliable biological assays especially complex array assays of many different samples.